Each papillomavirus is species specific and replicates persistently in a specific type of cutaneous or mucosal epithelium. The keratinocytes of the basal layer of the epithelium harbor a reservoir of replicating viral genomes and therefore are the ideal cells in which to study the mechanisms by which HPV establishes persistent infection. Papillomaviruses will only undergo their complete life cycle and generate progeny virus in the tissue culture equivalent of a stratified epithelium. This process requires establishing stably replicating viral DNA genomes in primary human keratinocytes and culturing them in three-dimensional skin equivalents. The aims of this project are to determine the mechanism of extrachromosomal replication and partitioning of the viral genome. We are studying the processes of genome establishment, persistence and genome amplification within the HPV life cycle. Most notably, this year we have determined that the papillomavirus E1 and E2 protein form subnuclear foci within infected cells and induce a DNA damage response that recruits cellular proteins required for DNA repair and synthesis to the foci. Thus, this defines an efficient mechanism for papillomaviruses to replicate their DNA without competition from host cell DNA replication.